Antibodies to Plasmodium falciparum

ABSTRACT

Antibodies which recognize and bind to histidine rich protein II of Plasmodium falciparum. These antibodies exhibit improved specificity and affinity for the antigen which provides enhanced sensitivity in immunoassays. Peptides useful for generation of the antibodies are also provided.

This is a division of application Ser. No. 08/250,309, filed May 27,1994, issued as U.S. Pat. No. 5,478,741 on Dec. 26, 1995 which is acontinuation of application Ser. No. 07/945.287, filed Sep. 11, 1992(abandoned).

FIELD OF THE INVENTION

The present invention relates to antibodies which recognize and bind toHRP-II, a histidine-rich protein of Plasmodium falciparum, assaysemploying these antibodies, and peptides for generating the antibodies.

BACKGROUND OF THE INVENTION

Plasmodium falciparum is the most pathogenic species of human malaria.The asexual parasites of this species in red blood cells produce severalproteins characterized by an unusually high content of the amino acidhistidine. One of these proteins is designated Histidine Rich Protein II(HRP-II). HRP-II is synthesized throughout the asexual stage of theparasite and is released from infected cells into the blood stream.

The HRP-II gene has been isolated and sequenced. The predicted aminoacid sequence includes 34% histidine, 37% alanine and 10% aspartic acid(Wellems and Howard. 1986. PNAS 83, 6065-6069). The HRP-II protein isalso unusual in that it contains many tandem repeats of the amino acidsequence Ala-His-His and a related sequence comprising (Wellems andHoward) about 80% of the sequence of the protein. The functions ofHRP-II and the other histidine-rich proteins are not yet known, butHRP-II is of particular interest for early detection of malarialinfection because it is transported out of infected cells into theextracellular medium where it can be detected in the blood of infectedpatients. Highly sensitive immunoassays for detection of HRP-II aretherefore desirable as a means for detecting malaria infection at anearly stage of the disease.

Antibodies specific for HRP-II are known in the art. Monoclonal antibodyMAb-87 and its subclones, raised to the intact HRP-II protein, have beendescribed by Howard, et al. (1986) J. Cell Biol. 103, 1269-1277; Rock,et al. (1987) Parasitology 95, 209-227 and Panton, et al. (1989) Molec.Biochem. Parasitology 35, 149-160. MAb-87 and its subclones are alsodisclosed in WO 89/01785 to Taylor. However, Mab-87 and its relatedmonoclonals are not suitable for diagnosis of malaria, as they arecapable of detecting a minimum of only 0.05% parasitemia in an enzymelinked immunosorbent assay (ELISA). Suppl. to Amer. J. Trop. Med. Hyg.1991. 45(3): 248-249. Polyclonal antibodies to a fragment of the HRP-IIprotein containing Ala-His-His and Ala-Ala-Asp repeats have beenreported by Knapp, et al. (1988. Behring Inst. Mitt. 82, 349-359).Wellems, et al. (1987. Cell 49, 633-642) have produced polyclonalantisera to a synthetic peptide having a similar amino acid sequence andthese antibodies bind to the HRP-II protein. Wellems and Howard disclosethese same polyclonal antisera and synthetic peptide in U.S. Pat. No.5,130,416. The foregoing authors and inventors also describe the use ofthe various anti-HRP-II antibodies to detect HRP-II antigen inimmunoassays such as Western blots and ELISA's.

SUMMARY OF THE INVENTION

The present invention provides antibodies which recognize and bind toHRP-II with increased specificity and affinity, thereby providingincreased sensitivity in immunoassays as compared to prior artanti-HRP-II antibodies. Monoclonal antibodies as well as polyclonalantisera were generated by immunization with a unique synthetic peptidecomprising the amino acid sequence (SEQ ID NO:1):

    Cys-Gly-Ala-His-His-Ala-His-His-Ala-Ala-Asp-Ala-His-His-Ala-Ala-Asp-Ala.

The N-terminal cysteine facilitates coupling of the peptide tomaleimide-derivatized carrier proteins through its --SH group. It isbelieved that the cysteine along with the glycine in the second positionserve as a spacer linkage between the HRP-II-like sequence and thecarrier protein which permits the peptide to assume a more authenticconformation for the production of antibodies. J. B. Rothbard et al. J.Exp. Med. 1984. 160: 208-221.

DETAILED DESCRIPTION OF THE INVENTION

The antibodies of the invention exhibit significantly improvedspecificity and affinity for HRP-II antigen in immunoassays as comparedto the antibodies described in the prior art. They therefore increasethe sensitivity of immunoassays and represent an important advance inthe ability of clinicians to detect the disease at an early stage. Theantibodies of the invention are capable of detecting as little as0.0008% parasitemia in plasma and 0.0002% parasitemia in whole blood,whereas an ELISA employing prior art antibodies could reliably detectonly parasitemias of 0.05% or greater (Schaeffler et al. 1991. Program &Abstracts of the 40th Annual Meeting of American Society of TropicalMedicine and Hygiene. Abstract #384. Amer. J. Trop. Med. Hyg. Suppl.45(3): 248-249). The percent parasitemia is determined by evaluating thenumber of infected red blood cells vs. uninfected red blood cells in astained blood film and is a reflection of the degree of infection. Theimproved antibodies of the invention are intended to encompass bothpolyclonal and monoclonal antibodies having the disclosed properties.

Taylor's prior art antibodies, as described in JCB (1986) 103, 1269,were produced in mice immunized with emulsified, purified red bloodcells infected with whole parasites. In contrast, the improvedantibodies of the invention were generated by immunizing mice with asynthetic peptide which is a modification of the peptide described byWellems and Howard in U.S. Pat. No. 5,130,416.

The immunogenic peptide has the amino acid sequence of SEQ ID NO:1:

    Cys-Gly-Ala-His-His-Ala-His-His-Ala-Ala-Asp-Ala-His-His-Ala-Ala-Asp-Ala.

wherein Cys represents the N-terminus of the peptide and Ala representsthe C-terminus. Residues 3-17 are a histidine-rich repeat sequencesimilar to segments of native HRP-II. The peptide may be synthesizedusing any of the synthetic methods known in the art, for example thesolid-phase method of Merrifield (1969. Advan. Enzymol. 32:221) or themodified solid-phase methods of Sheppard and Atherton (WO 86/03494)which are now automated. Chemical synthesis of the peptide is preferred.

An alternative method for producing the immunogenic peptide is byexpression of a recombinant oligonucleotide coding for the 18-mer.Methods for synthesizing an appropriate nucleic acid sequence, cloningit and expressing it in a transformed host cell are well known andwithin the ordinary skill in the art.

The cysteine residue at the N-terminus of the peptide facilitatescoupling of the peptide to an immunogenic carrier through the --SH groupof cysteine. Coupling of the peptide to an immunogenic carrier ispreferred for immunization, as coupling allows the small antigenicdeterminant peptide (a hapten) to elicit an antibody response. Commonlyused immunogenic carriers useful for coupling to haptens such as thepeptide of the invention are listed in Immunology, An IllustratedOutline by David Male, Gower Medical Publishing, 1986, pg. 31. Keyholelimpet haemocyanin (KLH) is the preferred immunogenic carrier forcoupling to the peptide to generate the antibody response.

The N-terminal Cys-Gly serves as a spacer linkage between the carrierand the peptide. While not wishing to be limited to any particularmethod by which the invention operates, Applicants believe that byproviding such a spacer linkage, the negative effects of the carrier onthe conformation of the peptide may be reduced, thus allowing thepeptide to assume a conformation more characteristic of a naturallyoccurring epitope of the HRP-II protein. This more authenticconformation may contribute to the ability of the peptide to elicit thesurprisingly high sensitivity antibodies of the invention.

Methods for coupling carriers to haptens through sulfhydryl groups areknown in the art and any of these are suitable for coupling the selectedcarrier to the immunogenic peptide. For example, see the couplingprotocols described in Current Protocols in Immunology, J. E. Coligan etal., eds., Greene Publishing Assoc. and Wiley Interscience, 1992,Chapter 9. Preferred reagents for coupling the sulfhydryl of cysteine toan amino group of a carrier protein are sulfosuccinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo SMCC) or SMCC andm-maleimidobenzoyl-N-hydroxysuccinimide (MBS) or sulfo-MBS because theyare specific for Cys-SH with essentially no secondary coupling reactionsat other amino acid residues. The most preferred coupling reagent issulfo SMCC, which may be used to conjugate the hapten to the peptideaccording to known methods such as S. Hashida et al. (1984) J. AppliedBiochemistry 56: 56-63. However, other coupling reagents such asglutaraldehyde may also be used with some secondary reaction at thehistidine residues.

The Ala residue at the C-terminus of the peptide serves to reduce thenegative charge density at the C-terminus and represents the next aminoacid which would be present in the native HRP-II protein.

Suitable methods for immunizing animals with synthetic peptides are alsowell known in the art. See Current Protocols in Immunology, supra. Ingeneral, an immunogenic amount of peptide/carrier conjugate is dissolvedor suspended in a physiological buffer, e.g., phosphate buffered saline(PBS), usually mixed with an adjuvant such as complete Freunds adjuvant.Animals are initially immunized with this mixture and thereafter boostedwith additional doses of peptide/carrier conjugate. The immunizationwith peptide/carrier conjugate is then repeated with an adjuvant such asincomplete Freunds adjuvant. At about 7 and 12 weeks after the initialimmunization the serum is generally tested using methods known in theart to determine the titer of antipeptide antibodies (e.g., reactivitywith the immunogen in an ELISA). Modifications and adjustments to thisbasic immunization protocol to obtain optimal antipeptide antibodytiters for any particular peptide/carrier conjugate are within theordinary skill in the art. If purified polyclonal antibody is desired,it may be isolated from the immune serum using well-established methods,such as separation on a peptide affinity column.

The spleen cells of an animal immunized with the immunogenic peptide mayalso be fused with murine myeloma cells for production of monoclonalantibodies using the method of Kohler and Milstein (1975. Nature 256,495-497) or other modifications of this method known in the art (Oi andHerzenberg. 1980. Selected Methods in Cellular Immunology, Mishell andShiigi, eds., pp. 351-372, W. H. Freeman, New York; Goding. 1986.Monoclonal Antibodies: Principles and Practice. Academic Press, SanDiego). The fused cells are cloned and screened for production of thedesired anti-peptide monoclonal antibody using immunological assays suchas ELISAs. If desired, purification of monoclonal antibody fromhybridoma culture supernatants or ascites fluid may be accomplishedusing methods known in the art, e.g., Protein G or peptide affinitycolumn chromatography.

The isolated polyclonal and monoclonal antibodies produced in responseto immunization with the peptide may be used in immunoassay protocols ofseveral types. The antibodies may be used intact or fragments may begenerated which are also capable of binding to the peptide and HRP-IIprotein (Fab or F(ab')₂). Intact antibodies as well as antigen bindingfragments thereof are intended to be encompassed by the presentinvention. While immunoassays can be performed using only polyclonalantibody reagents, in most cases monoclonal antibody or a combination ofpolyclonal and monoclonal antibodies are preferred. In general,antibodies or antigens in immunoassays are labeled by conjugation to adetectable label to facilitate detection of antigen/antibody binding byinclusion of the label in the binding complexes formed. As used herein,the term "label", "detectable label" and related terms are intended toencompass both the detectable label alone and, as described below,detectable labels associated with particles. Suitable labels and methodsfor conjugating them to proteins such as antibodies are well known.Directly detectable labels, which do not require additional reagents orreaction to be detected, include radioisotopes, fluorescent dyes andvisible absorbing dyes. Enzymes capable of reacting to produce coloredproducts are suitable indirectly detectable labels commonly used forconjugation to antibodies in specific binding assays. All of theforegoing labels are suitable for conjugation to the polyclonal andmonoclonal antibodies of the invention.

Particulate detectable labels are preferred for conjugation to theantibodies. Such particles include particles of polymers (e.g., latex orpolystyrene), sacs, liposomes, metallic sols (e.g., colloidal silver orcolloidal gold), other colloidal particles or polymeric dyes. To formthe particulate label, the particles are derivatized to include theselected detectable label, usually by formation of a chemical bond usingmethods known in the art for this purpose. Polymer particles, such aslatex particles, may also have the dye incorporated into the polymer. Inthe case of sacs and liposomes, the label may also be entrapped in thevesicle. The particle and its associated label may then be chemicallyconjugated to the antibody for use in specific binding assays.Alternatively, polymer particles, polymeric dyes and metal particles maybe coated with the antibody as described in U.S. Pat. No. 5,096,837. Thepreferred detectable labels for association with the present antibodiesare liposomes encapsulating an entrapped visible dye or other coloredparticles, with the antibody coupled to the surface of the liposome orparticle. Such liposome labels are described in U.S. Pat. No. 4,695,554.

The protocols for immunoassays using the antibodies are well known inthe art. For example, polyclonal or monoclonal antibodies according tothe invention or antigen binding fragments thereof may be employed insandwich assays for detecting HRP-II protein or in the knownmodifications and variations of sandwich assay protocols. Alternatively,the antibodies and antigen binding fragments thereof may be employed invarious competitive assay formats as are known in the art. The basics ofthese assay protocols are reviewed in Current Protocols in Immunology,supra. When used as a diagnostic for malaria infection, it is preferredthat the sample tested for the presence of HRP-II protein be eitherlysed or unlysed blood. However, other samples may be assayed

cultures, as well, for example, supernatants of infected cell extractsof P. falciparum parasites, serum, plasma, urine and cerebrospinalfluid.

Devices for performing specific binding assays, especially immunoassays,are known and can be readily adapted for use with the present monoclonaland polyclonal antibodies for detection of HRP-II. Solid-phase assays,in general, are easier to perform than heterogeneous assay methods suchas precipitation assays because separation of reagents is faster andsimpler. Solid-phase assay devices include microtiter plates,flow-through assay devices, dipsticks and immunocapillary orimmunochromatographic immunoassay devices as described in U.S. Pat. No.4,743,560; U.S. Pat. No. 4,703,017; U.S. Pat. No. 4,666,866; U.S. Pat.No. 4,366,241; U.S. Pat. No. 4,818,677; U.S. Pat. No. 4,632,901; U.S.Pat. No. 4,727,019; U.S. Pat. 4,920,046; U.S. Pat. No. 4,855,240; U.S.Pat. No. No. 5,030,558; U.S. Pat. No. 4,168,146. Most preferred areimmunocapillary assay devices which can be used as a dipstick, employingthe inventive monoclonal or polyclonal antibodies.

The preferred immunocapillary dipstick assay device is designed forconducting a sandwich immunoassay for HRP-II antigen. It comprises apiece of microporous absorbent material such as nitrocellulose laminatedto a plastic backing. In contact with the microporous material is astrip of a second absorbent material such as glass fiber, also laminatedto the plastic backing. Nitrocellulose is preferred for the firstmaterial because it allows immobilization of protein simply by applyingthe protein solution to the nitrocellulose and allowing it to beabsorbed. The second absorbent is in fluid communication with themicroporous material and assists in pulling the assay fluids through themicroporous absorbent. The second absorbent also absorbs the fluidswhich pass through the microporous material.

A monoclonal antibody according to the invention is immobilized on themicroporous absorbent in a position where it will not be directlyimmersed in the sample being tested. To perform the assay the portion ofthe microporous absorbent below the monoclonal antibody is contactedwith the sample such that the sample fluid is drawn up into it bycapillarity (wicking), thus bringing the sample into contact with theantibody and allowing binding between the antibody and any HRP-IIantigen which may be present in the sample. Thereafter, a solutioncontaining a visible dye-labeled polyclonal antibody according to theinvention is wicked up into the microporous absorbent into contact withthe monoclonal antibody/bound antigen complex such that the polyclonalantibody binds to the complexes through interaction with HRP-II.Optionally, a wash solution containing a mild detergent which will notdisrupt the liposomes (e.g., ZWITTERGENT) may be wicked into themicroporous absorbent after binding of the polyclonal antibody. Thedetectable dye label is then visualized in the area of immobilizedmonoclonal antibody.

In a preferred alternative embodiment of the dipstick assay device, apositive control area is included on the microporous absorbent in thevicinity of but distinct from the immobilized monoclonal antibody. Thepositive control may be HRP-II antigen, the immunogenic peptide or aderivative or analog thereof which also binds the antibodies of theinvention. The preferred positive control is HRP-II antigen immobilizedon nitrocellulose in an area contacted by the migrating sample fluidafter it contacts the area of immobilized monoclonal antibody.

The selected device and reagents for performing the immunoassay may bepackaged in the form of a kit for convenience. For example, such a kitmay include an appropriate assay device, antibody reagents, reagents fordevelopment of the assay such as buffers and, if needed, reagents fordetection of the chosen label.

The inventive antibodies may also be useful for reducing the risk of P.falciparum infection or treating such infection once established.Treatment may be accomplished by administering to an animal sufferingfrom malaria infection, preferably a human, a therapeutically effectiveamount of a pharmaceutical composition comprising a monoclonal orpolyclonal antibody according to the invention. Similar pharmaceuticallyacceptable compositions may be administered to an animal in a dosesufficient to increase immunity to subsequent P. falciparum infection.Alternatively, anti-idiotypic antibodies raised against the inventivemonoclonal or polyclonal antibodies may also-be administered in apharmaceutical composition as a vaccine against malaria infection.

The following experimental Examples are intended to illustrate certainfeatures and embodiments of the invention but are not to be consideredas limiting the scope of the invention as defined by the appendedclaims.

EXAMPLE 1

An 18-mer peptide having the amino acid sequence of SEQ ID NO:1 wasprepared using Fmoc-mediated synthetic chemistry with BOP and HOBTessentially as described by D. Hudson (1988. J. Org. Chem. 53: 617-624).A Milligen/Bio-Search EXCELL solid phase peptide synthesizer was used asrecommended by the manufacturer to perform the synthesis. The completedpeptide was deprotected and cleaved from the resin using Reagent R asrecommended by Milligen/BioSearch in their instructions. Crude peptidewas isolated by centrifuging the precipitate formed upon addition ofseven volumes of cold diethyl ether to combined Reagent R andtrifluoroacetic acid (TFA) washes of the resin. The precipitate waswashed with cold diethyl ether and subsequently dried in vacuo. Crudepeptide was further purified by chromatography on a C₁₈ -reverse phasematrix eluted with a gradient of 0.1% TFA in water (Buffer A) and 0.1%TFA in acetonitrile (Buffer B). The 18-mer peptide eluted at 33-34%Buffer B and was subsequently collected by lyophilizing pooled fractionscontaining the peptide. Conjugation of the purified peptide tosulfo-SMCC derivatized keyhole limpet hemocyanin (KLH) was done asdescribed by Rothbard, et al. (1984. J. Exp. Med. 160: 208-221) withminor modifications. Peptide conjugate was isolated from unconjugatedpeptide by chromatography on Sephadex G-75. The purified 18-merpeptide:KLH conjugate was subsequently used as an immunogen foreliciting antibodies to HRP-II.

Male BalbC/AnCrl mice (18-20 g) were used for immunization. A 2 mg/mlstock solution of the immunogenic peptide was prepared by reconstituting8 mg of lyophilized peptide in 4.0° ml of prewarmed PBS with vortexmixing until the powder dissolved. The stock solution was diluted 1:1with PBS and the 1 mg/ml solution was used for immunization. The firsttwo injections were given in the footpad and subcutaneously (100 μl ateach site of 1 mg/ml peptide:KLH with 100 μl Complete Freunds Adjuvant,Difco). This was followed by four intraperitoneal injections of 100 μlof peptide:KLH at approximately two day intervals. Anti-18-mer titerswere evaluated, and three of the four mice immunized responded well tothe peptide. Two intraperitoneal injections of 100 μl of peptide:KLHwere then given at approximately weekly intervals. After theseinjections, all four mice had some immune response to the peptide.Approximately three months later the mice were boosted with fourintraperitoneal injections of peptide:KLH. Based on reactivity with the18-mer peptide in an ELISA, one was selected for fusion of thesplenocytes with P3 myeloma cells according to a standard protocol suchas that described in "Current Protocols in Immunology," J. E. Coligan etal., eds., 1991, John Wiley & Sons, Chapter 2.

For screening immunized mice or hybridomas, ELISA plates were coatedovernight at 4° C. with 20 μg/ml of unconjugated 18-mer peptide, peptideconjugated to KLH (the immunogen) or peptide conjugated to BSA. Bleedsfrom immunized mice prior to splenocyte fusion or hybridoma culturesupernatants were diluted 1:10 in PBS-Tween buffer, followed by serialtwo-fold dilutions thereafter. The dilutions were incubated in theprepared ELISA plates for 1 hr at room temperature. Horseradishperoxidase conjugated goat anti-mouse immunoglobulin or rabbitanti-mouse IgG antibody was diluted in PBS-Tween and incubated with theELISA plates for 1 hr. O-phenylenediamine dihydrochloride enzymesubstrate was diluted in citrate-phosphate buffer and incubated with theELISA plates for 10 min. at room temperature. The absorbance of themedium was read at A₄₉₀.

Hybridomas resulting from the fusion were cloned and expanded based onreactivity with peptide:KLH in the ELISA. Clone MAL 18-27 was found tobe the only clone reactive with both the unconjugated peptide and theconjugated peptide, indicating that the antibody produced was specificfor the peptide rather than the KLH. MAL 18-27 was also shown to be18-mer specific, as it was the only clone tested which did not alsoreact with a 10-mer and/or a 21-mer in the ELISA. An isotyping assay ofclone MAL 18-27 showed the antibody produced to be an IgGlk isotype.

As it demonstrated the desired specificities, clone MAL 18-27 wasfurther subcloned. Two of these subclones, MAL 18-27.2.3.1 and MAL18-27.2.3.1.2, have been deposited with the American Type CultureCollection, Rockville, Md. under Accession Numbers and HB11111 andHB11112, respectively.

Monoclonal antibody MAL 18-27.2.3.1, the parent clone of MAL18-27.2.3.1.2, was tested in a solid phase dipstick immunoassay. Theimmunocapillary dipstick device comprised a strip of nitrocellulose(Schleicher & Schuell, Keene, New Hampshire) 7 mm wide and 25 mm longlaminated to a plastic backing strip. A strip of glass fiber (Gelman,Ann Arbor, Mich.) 7 mm wide and 5 cm long was also laminated to theplastic backing such that the glass fiber was in contact with andslightly overlapped the upper edge of the nitrocellulose. One μg ofmonoclonal antibody MAL 18-27.2.3.1 was spotted on the nitrocellulose inthe form of a line at approximately the midportion of the nitrocellulosestrip. Approximately 6 mm above the line, 0.17 μg of isolated HRP-IIantigen was spotted on the nitrocellulose in a dashed line. The HRP-IIantigen served as a positive control in the immunoassay.

The bottom edge of the nitrocellulose was placed in a shallow wellcontaining a 50 μl sample of lysed whole blood, serum or plasma suchthat the sample fluid was wicked up into the nitrocellulose and broughtinto contact with the immobilized antibody, allowing HRP-II antigenpresent in the sample to bind. A drop of a tracer reagent comprisingrabbit polyclonal antibody raised against the immunogenic peptideincorporated into liposomes with entrapped sulforhodamine B was placedin the well and wicked up into the nitrocellulose. The tracer reagentalso contacted and became bound to sample antigen complexed with theimmobilized monoclonal antibody. A wash reagent comprising 0.125%ZWITTERGENT 3-10 (Calbiochem, LaJolla, Calif.) was then wicked into thenitrocellulose.

In positive samples, the dye was visible in a line at the midpoint ofthe nitrocellulose, indicating a positive assay. The dashed line 6 mmabove the midpoint also bound the labeled polyclonal antibody,indicating that the device and reagents were performing correctly.

One patient with 40 parasites/μl of plasma (corresponding to 0.0008%parasitemia) gave a clear 1+positive response in the dipstick assay. Twopatients with 2 parasites/100 white blood cells in whole blood samples(corresponding to 0.003% parasitemia) gave a very strong 3-4+ positiveresponse in the dipstick assay.

EXAMPLE 2

The titer of monoclonal antibody MAL 18-27.2.3.1 to the peptide antigenwas compared to Mab 87-2G12 (WO 89/01785) as a measure of antibodyspecificity. Each well of a 96-well microtiter plate was coated with 50μl of a 20 μg/ml solution of peptide antigen in 0.1M carbonate buffer,pH 9.6 and incubated at 37° C., covered, overnight. The wells werewashed five times with 200 μl of PBS-0.05% Tween 20 buffer. Protein Gpurified monoclonal antibodies were serially diluted into the wells (50μl/well), starting at a protein concentration of 20 μg/ml. Theantibodies were allowed to bind to the immobilized antigen at 37°,covered for 1 hr and the wells were washed five times with 200 μl ofPBS-0.05% Tween 20 buffer.

Horse radish peroxidase (HRP) labeled goat anti-mouse Ig (OrganonTeknika, Durham, N.C.) was added at a 1:8,000 dilution in PBS-0.05%Tween 20 buffer, 50 μl/well, and incubated 1 hr. at 37° C., covered. Thewells were washed as above and 50 μl of O-phenylenediaminedihydrochloride (the enzyme substrate) were added for 10 min. at roomtemperature. The enzyme reaction was quenched by adding 50 μl/well of4.5 M H₂ SO₄.

The titer was calculated as the antibody concentration which gave anOD₄₉₀ of 1.00. The Mab 87-2G12 resulted in an OD reading of about 0.42at 20 μg/ml, indicating that a titer of greater than 20 μg/ml would benecessary for an OD reading of 1.00. The inventive monoclonal antibodyMAL 18-27.2.3.1, in contrast, had a titer of 1.79 μg/ml and thereforeexhibits a significantly higher specific activity for the peptideantigen. That is, to determine a fixed amount of peptide with anintensity of 1.00 OD units, only 1.79 μg/ml of MAL 18-27.2.3.1 isrequired as compared to more than 20 μg/ml of Mab 2G12.

In a second comparison, MAL 18-27.2.3.1 was tested against Mab 87-1E1(WO 89/01785) for antigen titer. The wells were coated with 50 μl/wellof Protein G purified monoclonal antibody (10 μg/ml in 0.1M carbonatebuffer) at 4° C. overnight. The plates were washed five times on aBiotek EL403 automated 96 well washer. The peptide antigen was seriallydiluted into the coated wells of the microtiter plates (50 μl/well,starting at 10 μg/ml) and incubated at 37° C. for 1 hr., covered, withshaking. The plates were again washed five times on the Biotek washer.

10 μg/ml solutions of the detector antibodies were prepared in PBS-0.05%Tween 20 buffer and 50 μl/well was added to the antigen captured plates.The plates were incubated 1 hr. at 37°, covered, with shaking. To ensurethat both ELISA's were optimized to their maximum detection ability, amonoclonal detector antibody was used for assay of Mab 87-1E1 but therabbit polyclonal detector antibody described in Example 1 was used toassay MAL 18-27.2.3.1.

Using Mab 87-1E1 as the capture antibody, about 144 μg/ml of peptideantigen was required to give an average OD reading of 1.0. Using MAL18-27.2.3.1 as the capture antibody, only 0.8-1.2 μg/ml of peptideantigen was required for an average OD reading of 1.0. These resultsdemonstrate a significantly higher sensitivity in the assay using theinventive antibody.

EXAMPLE 3

Monoclonal antibody MAL 18-27.2.3.1.2 was clinically tested incomparison with a commercially available diagnostic test (QBC, BectonDickinson Advanced Diagnostics, Baltimore, Md.) as a diagnostic for P.falciparum infection using the dipstick assay format previouslydescribed. QBC results were confirmed by microscopic analysis of thinfilms. All tests were performed on anticoagulated venous blood samplesaged 2-24 hrs. and kept under refrigeration. Tests were also performedafter four days on three samples.

The twelve samples which tested positive by QBC and thin films alsotested positive in the dipstick assay. One of these samples was from apatient exhibiting only gametocytes. The dipstick assay was positive forparasitemias below 0.1%, including one parasitemia which showed only 3parasites on the thin film.

One patient followed for five days showed an initial parasitemia of 4%which progressed to rare trophoblasts, then rare gametocytes on the thinfilms. However, at each stage the dipstick assay was clearly positive.

Twenty-one patient samples which tested negative by QBC and thin filmswere tested with the dipstick assay. Three of these patients, withproven P. falciparum malaria, had been treated with intravenous quininetherapy. After three days their QBC and films remained negative, but thepatients tested positive with the dipstick assay for as long as sixdays.

These results demonstrate that antibodies raised to the 18-mer peptidehave increased specificity and sensitivity in clinical tests as comparedto anti-P. falciparum antibodies currently used in diagnostics.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CysGlyAlaHisHisAlaHisHisAlaAlaAspAlaHisHisAlaAla                              151015                                                                        AspAla                                                                        __________________________________________________________________________

What is claimed is:
 1. A method of making an antibody comprising:a)immunizing an animal with a peptide consisting of SEQ ID NO:1 coupled toan immunogenic carrier; b) preparing a hybridoma which produces amonoclonal antibody having the specificity and sensitivity of monoclonalantibodies produced by ATCC Accession No. HB 11111 or ATCC Accession No.HB 11112, and; c) isolating the monoclonal antibody produced by thehybridoma.
 2. The method of claim 1 wherein the animal is immunized withthe peptide dissolved or suspended in a physiological buffer.
 3. Themethod of claim 1 wherein the animal is immunized with SEQ ID NO:1coupled to keyhole limpet haemocyanin.
 4. The method of claim 1 whereinthe monoclonal antibody is isolated by binding to Protein G or SEQ IDNO:1.
 5. A method of making a hybridoma comprising:a) immunizing ananimal with a peptide consisting essentially of SEQ ID NO:1 coupled toan immunogenic carrier; b) obtaining spleen cells from the immunizedanimal, and; c) preparing a hybridoma from the spleen cells whichproduces a monoclonal antibody having the specificity and sensitivity ofmonoclonal antibodies produced by ATCC Accession No. HB 11111 or ATCCAccession No. HB
 11112. 6. The method of claim 5 wherein the animal isimmunized with SEQ ID NO:1 coupled to keyhole limpet haemocyanin.
 7. Themethod of claim 5 wherein the hybridoma is prepared by fusing the spleencells of the immunized animal with mouse myeloma cells.
 8. The method ofclaim 5 wherein the animal is immunized with the peptide dissolved orsuspended in a physiological buffer.